1 Rapid apoptosis induction by IGFBP-3 involves an IGF -independent nucleo- mitochondrial translocation of RXRα/Nur77 Kuk-Wha Lee 1 , Liqun Ma 1 , Xinmin Yan 1 , Bingrong Liu 1 , Xiao-kun Zhang 2 & Pinchas Cohen 1* 1 Division of Pediatric Endocrinology, Mattel Children's Hospital at UCLA, David Geffen School of Medicine, Los Angeles, CA, USA 2 Cancer Center, The Burnham Institute, La Jolla, CA, USA Running title: IGFBP-3 translocates RXRα/Nur77 *To whom correspondence may be addressed. Tel: (310) 206-5844. Fax: (310) 206-5843. E-mail: [email protected]SUMMARY Insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis by its ability to bind IGFs as well as its IGF-independent effects involving binding to other molecules including the retinoid X receptor-α (RXRα). Here we describe that in response to IGFBP-3, the RXRα binding partner nuclear receptor Nur77, rapidly undergoes translocation from the nucleus to the mitochondria, initiating an apoptotic cascade resulting in caspase activation within 6 hours. This translocation is a type 1 IGF receptor signalling-independent event as IGFBP-3 induces Nur77 translocation in R- cells. IGFBP-3 and Nur77 are additive in inducing apoptosis. GFP-Nur77 transfection into RXRα WT and KO MEFs and subsequent treatment with IGFBP-3 shows that RXRα is required for IGFBP-3 induced Nur77 translocation and apoptosis. Addition of IGFBP-3 to 22RV1 cell lysates enhanced the ability of GST- RXRα to “pull-down” Nur77, and overexpression of IGFBP-3 enhanced the accumulation of mitochondrial RXRα. This unique non-genotropic nuclear pathway supports an emerging role for IGFBP-3 as a novel, multi-compartmental signaling molecule involved in induction of apoptosis in malignant cells. JBC Papers in Press. Published on February 24, 2005 as Manuscript M412757200 Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on June 5, 2018 http://www.jbc.org/ Downloaded from
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Rapid apoptosis induction by IGFBP-3 involves an IGF -independent nucleo-mitochondrial translocation of RXRα/Nur77
1Division of Pediatric Endocrinology, Mattel Children's Hospital at UCLA, DavidGeffen School of Medicine, Los Angeles, CA, USA2Cancer Center, The Burnham Institute, La Jolla, CA, USA
Running title: IGFBP-3 translocates RXRα/Nur77
*To whom correspondence may be addressed. Tel: (310) 206-5844. Fax: (310)206-5843. E-mail: [email protected]
SUMMARY
Insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis by its
ability to bind IGFs as well as its IGF-independent effects involving binding to
other molecules including the retinoid X receptor-α (RXRα). Here we describe
that in response to IGFBP-3, the RXRα binding partner nuclear receptor Nur77,
rapidly undergoes translocation from the nucleus to the mitochondria, initiating
an apoptotic cascade resulting in caspase activation within 6 hours. This
translocation is a type 1 IGF receptor signalling-independent event as IGFBP-3
induces Nur77 translocation in R- cells. IGFBP-3 and Nur77 are additive in
inducing apoptosis. GFP-Nur77 transfection into RXRα WT and KO MEFs and
subsequent treatment with IGFBP-3 shows that RXRα is required for IGFBP-3
induced Nur77 translocation and apoptosis. Addition of IGFBP-3 to 22RV1 cell
lysates enhanced the ability of GST- RXRα to “pull-down” Nur77, and
overexpression of IGFBP-3 enhanced the accumulation of mitochondrial RXRα.
This unique non-genotropic nuclear pathway supports an emerging role for
IGFBP-3 as a novel, multi-compartmental signaling molecule involved in
induction of apoptosis in malignant cells.
JBC Papers in Press. Published on February 24, 2005 as Manuscript M412757200
Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
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Fig. 1. Rapid Activation of Apoptosis by IGFBP-3. A, Time course apoptosisinduction of Mouse Embryonic Fibroblasts (MEFs) after treatment with 1 µg/ml ofIGFBP-3. Apoptosis induction was quantitated by fluorometric measurement ofactivated caspase 3/7. Values are represented as percent of Serum Free. B,Time course of human glioblastoma line A172 apoptosis induction aftertreatment with 1 µg/ml of IGFBP-3. Apoptosis induction was quantitated byfluorometric measurement of activated caspase 3/7. Values are represented aspercent of Serum Free. C, Time course of human prostate cancer line 22RV1apoptosis induction after treatment with 1 µg/ml of IGFBP-3. Apoptosis inductionwas quantitated by fluorometric measurement of activated caspase 3/7. Valuesare represented as percent of Serum Free. ** = p<0.005 relative to Serum Freeconditions.
Fig. 2. IGFBP-3 induces nucleo-mitochondrial translocation of Nur77. A,Indirect immunofluorescent confocal microscopy of 22RV1 cells after treatmentwith IGFBP-3. Nur77 is labeled in red. Nuclei are labeled in blue. Note rapidappearance of cytoplasmic Nur77. SFM, Serum Free Medium B, Westernimmunoblot of subcellular fractions of 22RV1 prostate cancer cells aftertreatment with 1mcg/ml IGFBP-3, probed for Nur77. PARP, and Hsp60 areloading controls and show purity of the nuclear fraction. C, 22RV1 mitochondrialfraction isolated after treatment with 1mcg/ml IGFBP-3. Membrane was probedwith PARP, PMP70, and Cathepsin S to show purity of the mitochondrial fraction.
Fig. 3. Rapid Mitochondrial Accumulation of Nur77by IGFBP-3 occurs via a type1 receptor independent mechanism. A, Type 1 IGF receptor disrupted mouseembryonic fibroblasts (R- MEFs) were treated with IGFBP-3 (1 µg/ml) for theindicated times and apoptosis was measured in a fragmented DNA/histoneELISA assay. B, The mitochondrial fraction was analyzed for expression of Nur77by Western blotting. To demonstrate the purity of the mitochondrial fraction,expression of mitochondrial-specific protein Hsp60 and nuclear-specific proteinpoly(ADP-ribosyl) polymerase (PARP), as well as peroxisomal PMP70 andlysosomal cathepsin S is shown. A representative of 3 separate experiments isshown. Values are represented as percent of Serum Free. ** = p<0.005 relativeto Serum Free conditions.
Fold Increase from baselineCytoplasmic cytochrome c
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Fold Increase from baseline Caspase 3/7 Activation
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Fig. 4. Additive effects on apoptosis by IGFBP-3 and Nur77. A, Release ofcytoplasmic cytochrome c by IGFBP-3. Values are expressed as fold increasefrom baseline derived from densitometric analysis of western immunoblots ofmitochondria-depleted cytoplasmic fractions probed with cytochrome c. Valueswere normalized for loading with β-actin. **P<0.005 relative to time 0. B, Caspaseactivation post transient transfection of IGFBP-3, Nur77; alone and incombination. Values are nomalized to β-galactosidase expression to adjust fortransfection efficiency. **P<0.005 relative to vector alone, and also forcombination compared to Nur77 alone. C, Western immunoblot ofoverexpressing transiently transfected cells whole cell lysates.
Fig. 5. RXRa is required for IGFBP-3-induced Nur77 translocation. A,Differential subcellular localization of Nur77 to IGFBP-3 in RXRα+/+ and RXRα-/-
cells. Values are expressed as fold increase from baseline derived from Nur77densitometric analysis of western immunoblots of nuclear fractions of F9 cellsafter treatment with IGFBP-3. Values are normalized to nuclear PARP to adjustfor loading. **P<0.005 relative to no treatment. B, Confocal microscopy of F9 cellstransfected with GFP-Nur77. Note extranuclear appearance of GFP-Nur77 aftertreatment with IGFBP-3. C, Caspase activation post transfection of Nur77expression vector. **P<0.005 relative to control vector alone. D, GST-RXRα pull-down of 22RV1 cell lysates treated with IGFBP-3. IGFBP-3 enhances the ability ofRXRα and Nur77 to physically associate. CCRF-CEM nuclei was used as apositive control for Nur77 protein expression E, Overexpression of IGFBP-3enhances mitochondrial RXRα accumulation. Mitochondrial fraction of 22RV1cells transiently transfected with IGFBP-3 expression vector. Fraction isimmunoblotted with anti-RXRα.